Interactions of Cbl with Grb2 and phosphatidylinositol 3'-kinase in activated Jurkat cells.
T-cell receptor (TCR) cross-linking increases tyrosine phosphorylation of multiple proteins, only a few of which have been identified. One of the most rapidly tyrosine-phosphorylated polypeptides is the 120-kDa product of the proto-oncogene c-cbl, a cytosolic and cytoskeletal protein containing multiple proline-rich motifs that are potential binding sites for proteins containing ... Src homology 3 (SH3) domains. We report here that in cultured Jurkat T cells, Cbl is coprecipitated with antibody against the adapter protein Grb2. Upon activation of Jurkat T cells via the TCR-CD3 complex, we find that high-affinity binding of Cbl requires the N-terminal SH3 domain of GST-Grb2 fusion protein but after cross-linking of the TCR-CD3 and CD4 receptors, Cbl binds equally to its SH2 domain. Grb2 antisera also precipitated p85 from serum-starved cells, while TCR activation increased p85 and tyrosine-phosphorylated Cbl but not Cbl protein in Grb2 immunocomplexes. Phosphatidylinositol (PI) 3-kinase activity was immunoprecipitated from serum-starved cells with Cbl and to a lesser extent with Grb2 antisera, and TCR cross-linking increased this activity severalfold. The PI 3-kinase activity associated with Cbl amounted to 5 to 10% of the total cellular activity that could be precipitated by p85 antisera. The Ras exchange factor Son-of-sevenless 1 (Sos-1) was not found in anti-Cbl immunoprecipitates from activated cells, and Cbl was not detectable in anti-Sos-1 precipitates, supporting the likelihood that Sos-Grb2 and Cbl-Grb2 are present as distinct complexes. Taken together, these data suggest that Cbl function in Jurkat T cells involves its constitutive association with Grb2 and its recruitment of PI 3-kinase in response to TCR activation.
Mesh Terms:
Adaptor Proteins, Signal Transducing, Antigens, CD3, Base Sequence, Blotting, Western, GRB2 Adaptor Protein, Lymphocyte Activation, Membrane Proteins, Molecular Sequence Data, Phosphatidylinositol 3-Kinases, Phosphotransferases (Alcohol Group Acceptor), Precipitin Tests, Protein Binding, Proteins, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-cbl, Receptors, Antigen, T-Cell, Recombinant Fusion Proteins, Signal Transduction, Son of Sevenless Proteins, T-Lymphocytes, Ubiquitin-Protein Ligases
Adaptor Proteins, Signal Transducing, Antigens, CD3, Base Sequence, Blotting, Western, GRB2 Adaptor Protein, Lymphocyte Activation, Membrane Proteins, Molecular Sequence Data, Phosphatidylinositol 3-Kinases, Phosphotransferases (Alcohol Group Acceptor), Precipitin Tests, Protein Binding, Proteins, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-cbl, Receptors, Antigen, T-Cell, Recombinant Fusion Proteins, Signal Transduction, Son of Sevenless Proteins, T-Lymphocytes, Ubiquitin-Protein Ligases
Mol. Cell. Biol.
Date: Jul. 01, 1995
PubMed ID: 7791764
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